Enhanced Generation of Cytotoxic T Lymphocytes by Heat Shock Protein 70 Fusion Proteins Harboring Both CD8(+) T Cell and CD4(+) T Cell Epitopes

Heat shock protein 70 (Hsp70) can be a potent carrier of antigens because it is effectively delivered to antigen presenting cells (APCs) and activates innate immunity. To induce a potent cytotoxic T lymphocyte (CTL) response, a Hsp70 fusion protein harboring both CD8(+) and CD4(+) T cell epitopes was developed based on the recent understanding of the importance of the role of CD4(+) T cells in inducing the CTL response following vaccination. OVA(257-264) (pepI) and OVA(323-339) (pepII) were selected as the CD8(+) and CD4(+) T cell epitope of a model antigen, ovalbumin (OVA), respectively. Hsp70 and its fusion proteins, Hsp70-pepI, pepII-Hsp70, Hsp70-pepII and pepII-Hsp70-pepI, were developed. pepII-Hsp70 and pepII-Hsp70-pepI were effectively presented on MHC class II of macrophages compared with Hsp70-pepII, suggesting that pepII conjugation to the N-terminus of Hsp70 is better than the C-terminus for more effective MHC class II antigen presentation. Immunization with pepII-Hsp70-pepI resulted in a higher CTL activity than immunization with the mixture of Hsp70-pepI and pepII-Hsp70. Furthermore, pepII-Hsp70-pepI exhibited a greater antitumor effect in the mice bearing EG7 tumor cells than the physical mixture of Hsp70-pepI and pepII-Hsp70. In addition, immunization with the DCs pulsed with the fusion proteins also suggested that APCs which present both pepI and pepII can induce the highest CTL generation. These results demonstrated that Hsp70 fusion protein harboring both pepI and pepII is a useful option for Hsp70-based antigen delivery systems.